Recommended protocol for separation and purification of recombinant enterokinase and protein of interest
March 9, 2022
Recombinant enterokinase is designed to cleave recombinant expression of fusion proteins and is divided into two types: rEK-B and rEK-H, which do not contain His-6 and contain His-6 recombinant tags.
Yaxin Bio currently produces rEK-B. It does not contain a His-tag, and its relative activity is high, about 20,000 Unit/mg pro. According to the activity definition, 1 Unit can cut 0.5mg of the target protein, and the recombinant intestine is cut. The amount of the kinase relative to the protein of interest is one in ten thousand (0.01%). Therefore, in further experiments, the effect on downstream experiments may be disregarded, suggesting that it may not be purified.
If you really need to remove rEK, the recommended removal method is as follows:
If affinity chromatography is used, a trypsin inhibitor affinity gel can be utilized. Since enterokinase is a serine protease, trypsin inhibitors can bind to enterokinase for purification purposes. It is recommended to separate and remove trace amounts of enterokinase by ion exchange chromatography.
Enterokinase binds to DEAE gel under conditions of pH 8.0 and can be eluted in 0.15 M NaCl. The two proteins can be separated by different conditions of binding of the protein of interest to the intestinal kinase charge and binding to the DEAE gel and elution.